FIGURE 6.

PINK1 knockdown elicits mitochondrial superoxide upstream of mitochondrial fragmentation and autophagy in PINK1-deficient lines. A, epifluorescence analysis of MitoSOX, a cell-permeable red fluorescent mitochondrial superoxide indicator, in a control stable cell line (Ctrl, left panel) and two PINK1 knockdown cell lines (right panels). Nuclei were counterstained with DRAQ5 (blue). Scale bar: 100 μm. B, bar graph shows compiled means ± S.E. from three independent experiments with increases in the average MitoSOX fluorescence per cell normalized to vector control cell line (, p < 0.05 versus vector control cell line; n = 200-300 cells analyzed/condition). C and D, quantification of mitochondrial interconnectivity (C) and mitochondrial elongation (D) in PINK1 knockdown clonal cell lines treated with catalase, MnTBAP, or vehicle (untreated) for 24 h. (Representative of three independent experiments; , p < 0.05 versus control cell line; n = 25-35 cells analyzed per condition.) E, representative epifluorescence micrograph of a PINK1 knockdown cell line (A4) transiently expressing GFP-LC3 treated in the presence or absence of antioxidant MnTBAP. Note that MnTBAP suppresses autophagy in PINK1 knockdown cells. F, the average cellular number of GFP-LC3 puncta in control or PINK1shRNA cells transiently expressing GFP-LC3 in the presence or absence of indicated antioxidants. (, p < 0.05 versus untreated control cell line; , p < 0.0001 versus respective untreated clones; representative bar graph shows means ± S.E., n = 25-40 cells quantified/condition).